Abstract: The current roadblocks to hematopoietic stem cell (HSC) therapies include the rarity of matched donors for bone marrow transplant, engraftment failures, common shortages of donated blood, and the inability to expand HSCs ex vivo in large numbers. These major obstacles would cease to exist if an extensive, bankable, inexhaustible, and patient-matched supply of blood were available. The recent validation of hemogenic endothelium has introduced new possibilities in hematopoietic stem cell therapy. The ability of the arterial endothelium from embryonic vascular sites to produce definitive life-long HSCs!n absence of differentiation, during a defined time window, suggests that if recapitulated in culture, the clinical applications would prove quite powerful. The project's objective is to engineer mature human endothelium for in vitro production of hematopoietic stem cells. The central hypothesis is that the governing programs of hematopoietic emergence from the endothelium can be activated in mature endothelium for HSC production. The hypothesis is based on the changing expression of key transcription factors active in hemogenic endothelium and silent in mature endothelium; and vice versa with the later maintaining endothelial quiescence. The innovative approach employs the co-option of tissue specific factors to de-differentiate mature cells into their slightly more primitive states as an alternative to adopting the pluripotent reprogramming blueprint that creates universal plasticity. This allows maintenance of tissue identity but with increased function and tissue specific plasticity, while minimizing malignant risk and alternate cell fate decisions. In the case of hemogenic endothelium, it will allow for recapitulation of hemogenic function. Engineering endothelium into large-scale hematopoietic factories can provide substantial numbers of pure hematopoietic stem cells for clinical use. Higher numbers of cells, and the ability to grow cells from matched donors (or the patients themselves) will increase engraftment and decrease rejection of bone marrow transplantation. In addition, the ability to program mature lineage restricted cells into more primitive versions of the same cell lineage will capitalize on cell renewal properties while minimizing malignancy risk. Public Health Relevance: The proposed project is relevant to public health because it will allow the eventual banking of umbilical cord endothelium instead of cord blood to provide a patient matched inexhaustible supply of pure hematopoietic stem cells for the entire life of the patient. The innovative approach of re-engineering tissue specific dedifferentiation will accelerate the pace of discovery and translation to human disease in direct agreement with the mission of the NIH.